Bacterial chemotaxis towards the extracellular products of
the toxic phytoplankton Heterosigma akashiwo
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Citation
Seymour, Justin R., Tanvir Ahmed, and Roman Stocker.
“Bacterial chemotaxis towards the extracellular products of the
toxic phytoplankton Heterosigma akashiwo.” Journal of Plankton
Research 31.12 (2009): 1557 -1561.
As Published
http://dx.doi.org/10.1093/plankt/fbp093
Publisher
Oxford University Press
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Author's final manuscript
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Thu May 26 09:53:39 EDT 2016
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http://hdl.handle.net/1721.1/60930
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http://creativecommons.org/licenses/by-nc-sa/3.0/
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SHORT COMMUNICATION
Bacterial chemotaxis towards the
extracellular products of the toxic
phytoplankton Heterosigma akashiwo
JUSTIN R. SEYMOUR1,2*, TANVIR AHMED1 AND ROMAN STOCKER1
1
RALPH M. PARSONS LABORATORY, DEPARTMENT OF CIVIL AND ENVIRONMENTAL ENGINEERING, MASSACHUSETTS INSTITUTE OF TECHNOLOGY, 77
2
MASSACHUSETTS AVE, CAMBRIDGE, MA, USA AND SCHOOL OF BIOLOGICAL SCIENCES, FLINDERS UNIVERSITY, GPO BOX 2100, ADELAIDE, SOUTH AUSTRALIA
5001, AUSTRALIA
*CORRESPONDING AUTHOR: justins@mit.edu
Received July 22, 2009; accepted in principle September 3, 2009; accepted for publication September 10, 2009
Corresponding editor: William Li
Marine bacteria exhibit positive chemotactic responses to the extracellular exudates of the toxic phytoplankton Heterosigma akashiwo. In the environment, this will support bacteria – algae associations with potential implications for harmful algal bloom dynamics.
The occurrence of harmful algal blooms (HABs),
caused by toxic phytoplankton, has detrimental environmental, economic and human health effects within
many aquatic habitats (Horner et al., 1997).
Understanding the environmental conditions that
control the development and decline of HABs has consequently become an important element of ecosystem
management within heavily affected environments.
Potentially significant links between HAB dynamics and
heterotrophic bacteria have been reported, with various
species of aquatic bacteria implicated in both the promotion and inhibition of HABs (Kim et al., 1998;
Skerratt et al., 2002; Liu et al., 2008a, b). This has
revealed the unseen complexity of HAB ecology, as well
as arousing interest in the potential application of algicidal bacteria as biocontrols to terminate HABs (Kim
et al., 2007).
The raphidophyte phytoplankton Heterosigma akashiwo
is a toxic species that has been implicated in several
major fish kills worldwide (Honjo, 1993). The mechanism behind H. akashiwo toxicity is still unresolved, but
increases in the toxic effects of this species have been
linked to ecological interactions with heterotrophic bacteria (Carrasquero-Verde, 1999). Furthermore, heterotrophic bacteria can both enhance (Liu et al., 2008a, b)
and inhibit the growth of H. akashiwo (Kim et al., 1998;
Lovejoy et al., 1998; Yoshinaga et al., 1998; Liu et al.,
2008a, b), indicating that bacterial-algal interactions
play a fundamental role in the ecology and environmental impact of this toxic species. Behavioural
responses, including localized bacterial clustering
around H. akashiwo cells (Lovejoy et al., 1998), forming
specific algal – bacterial consortia (Carrasquero-Verde,
1999), have been predicted to be an important element
of these interactions. In this study, we examined
whether the chemical products of H. akashiwo act as
chemoattractants for three environmentally relevant
strains of marine bacteria, to elucidate how behavioural
doi:10.1093/plankt/fbp093, available online at www.plankt.oxfordjournals.org
# The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
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strength 2216 marine broth (Miller et al., 2004) to midexponential phase, centrifuged at 1500 g for 5 min and
washed in ASW.
A microfluidic chemotaxis assay, described in detail
elsewhere (Seymour et al., 2008), was used to study the
chemotactic response of bacteria to the extracellular
products of H. akashiwo. The microfluidic device consisted of a 45 mm long, 3 mm wide and 50 mm deep
microchannel (Fig. 1A). Two in-line inlet ports were
used to simultaneously introduce bacteria (inlet 1) and
chemoattractants (inlet 2) into the channel, at a constant
flow rate using a syringe pump. Inlet 2 led to a 100 mm
wide microinjector, which produced a 300 mm wide
band of chemoattractants in the centre of the channel.
Bacteria were advected along either side of the chemoattractant band, until flow in the channel was
stopped by turning off the syringe pump. At this point,
the chemoattractant band was “released”, spreading laterally at a rate set by molecular diffusion. The chemotactic response of the bacteria was then measured.
Positions of bacteria across the microchannel were
visualized using an inverted microscope (Nikon
TE2000e) and “movies” (200 frames) were recorded at
32 frames s21 at 2 min intervals using a CCD camera
(PCO 1600, Cooke, Romulus, MI, USA). Image analysis software (IPlab, BD Biosciences Bioimaging, MD,
USA) was used to determine the mean distribution of
bacteria across the channel (x direction). To compare
the strength of chemotactic accumulation between
experiments, cell concentrations were normalized with
respect to the mean concentration in the outermost
right 600 mm of the microchannel. A chemotactic index
IC, defined as the mean of the normalized cell
responses may catalyse the ecological coupling between
this toxic phytoplankton species and associated bacteria.
Axenic cultures of Heterosigma akashiwo (CCMP452)
were grown in O3 medium (McIntosh and Cattolico,
1978) to mid-exponential phase. The extracellular exudates of H. akashiwo were obtained using the method of
Bell and Mitchell (Bell and Mitchell, 1972). Cells were
centrifuged for 5 min at 500 g and then gently filtered
through sterile 0.2 mm membrane filters (Millipore).
The culture filtrate (exudates) was then employed as a
chemoattractant substrate for three environmentally
relevant marine bacterial isolates. These included
(i) Pseudoalteromonas haloplanktis (ATCC700530), a species
previously shown to increase the toxicity of H. akashiwo
(Carrasquero-Verde, 1999); (ii) Vibrio alginolyticus
(12G01), a species shown to be inhibited by H. akashiwo
(Oda et al., 1997); and (iii) Silicibacter sp. (TM1040), a
species known to establish close spatial associations with
other phytoplankton species (Miller et al., 2004).
Prior to experiments, bacteria were grown and prepared for chemotaxis experiments according to previously established protocols (Malmcrona-Friberg et al.,
1990; Mitchell et al., 1996; Miller et al., 2004).
Pseudoalteromonas haloplanktis was grown in 1% Tryptic
Soy Broth supplemented with 400 mM NaCl. Cells
were harvested at mid-exponential growth phase and
diluted 1:20 in artificial seawater (ASW) solution, before
being starved at room temperature for 72 h (modified
from Mitchell et al., 1996). Vibrio alginolyticus was grown
in Vibrio Nine Salt Solution (VNSS) to mid-exponential
phase, centrifuged at 1500 g for 5 min and washed in
Nine Salt Solution (NSS) (Malmcrona-Friberg et al.,
1990). Silicibacter sp. (TM1040) was grown in half-
Fig. 1. (A) Schematic diagram of the microfluidic chemotaxis assay. Bacteria and chemoattractants were injected into the channel via inlets 1
and 2, respectively. The channel outlet is denoted by 3. (B) Swimming trajectories of V. alginolyticus bacteria within a band of Heterosigma akashiwo
extracellular exudates, demonstrating accumulation of cells. Each white path is the trajectory of a single bacterium. Trajectories were obtained
from a 6.2 s long movie recorded at 32.4 frames per second. Note, the orientation of (B) has been rotated 908 from (A). In both panels, the area
bound by the grey dotted box corresponds to the approximate initial position of the band of injected H. akashiwo exudates.
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CHEMOTAXIS TO H. AKASHIWO EXUDATES
Table I: Mean values of the chemotactic index
IC for heterotrophic bacteria responding to the
extracellular exudates of Heterosigma akashiwo
at different time points after flow in the
microfluidic channel was stopped
Time (min)
P. haloplanktis
TM1040
V. alginolyticus
2
4
6
8
10
0.85 + 0.1
0.95 + 0.2
0.95 + 0.2
1.04 + 0.2
0.94 + 0.01
3.6 + 1.2
3.3 + 0.9
2.4 + 0.4
1.9 + 0.2
1.5 + 0.5
1.5 + 0.4
2.0 + 0.4
2.9 + 0.5
3.3 + 0.6
4.6 + 1.3
Mean and standard deviation values were calculated from triplicate
experiments.
concentration over the central 600 mm of the microchannel, corresponding to the position of the chemoattractant band, was also computed (Seymour et al.,
2008). IC ¼ 1 corresponds to a uniform cell distribution
(no chemotaxis), while strong chemotaxis is characterized by larger values of IC. Experiments were conducted in triplicate and the intensity, speed and
duration of the chemotactic responses were compared
between bacteria for each chemoattractant by comparing mean IC values using t-tests.
Different degrees of chemotactic response were exhibited by the three strains of heterotrophic bacteria
(Table I). On average, V. alginolyticus exhibited the strongest attraction to the H. akashiwo exudates, with cells
accumulating within the centre of the microfluidic
channel in correspondence with the position of the chemoattractant band (Fig. 1B). Within this central band,
cell densities exceeded background concentrations by
up to 6.5-fold (Fig. 2A). Vibrio alginolyticus attained a
maximum mean IC of 4.7 + 1.3 SD, 10 min after the
band of exudates was released. Silicibacter sp. TM1040
also exhibited a similar (P . 0.05) positive chemotactic
response (Fig. 2B), reaching mean IC ¼ 3.6 + 1.2.
While the strength of attraction was comparable, relative to V. alginolyticus, TM1040 reached maximum levels
of chemotactic attraction much more rapidly, with peak
cell concentrations within the chemoattractant band
occurring after 2 min (Table I). In contrast, P. haloplanktis
did not exhibit chemotactic attraction to the extracellular products of H. akashiwo (Fig. 2C), reaching a
maximum IC of only 1.0 + 0.2 (Table I). This response
by P. haloplanktis was significantly weaker (P , 0.05) than
the responses of V. alginolyticus and Silicibacter sp.
TM1040, and not distinguishable (P . 0.05) from the
control experiment, where O3 growth medium was
used as a chemoattractant. Alternatively, in the case of
V. alginolyticus and Silicibacter sp. TM1040, no positive
chemotactic response was exhibited in the control
Fig. 2. Representative experimental distributions of bacteria
across the width of the microfluidic channel at different times
following the injection of a band of Heterosigma akashiwo exudates. The
chemoattractant band was initially between x¼+150 mm, and t ¼ 0
corresponds to the release of the chemoattractant band. Bacterial
concentrations were normalized to the mean concentration measured
in the 600 mm closest to the outer right wall of the microchannel.
experiments (mean IC ¼ 1.1 + 0.2), confirming that the
significant (P , 0.05) chemotactic aggregation by V. alginolyticus and Silicibacter sp. TM1040 in the experimental
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The positive chemotactic behaviour exhibited here is in
line with these observations, but since no ecological
links between members of the Roseobacter clade and
H. akashiwo have previously been reported, the potential
implications of the chemotactic attraction observed here
remain open to further investigation.
The variability in the strength of chemotactic
response exhibited between bacterial strains tested here
could be driven by several factors. Differences in swimming speed and chemosensory capabilities will fundamentally influence how rapidly bacteria can exploit a
diffusing nutrient source. Chemosensory sensitivities are
also likely to be tuned to environmental parameters,
with bacteria inhabiting more oligotrophic habitats,
such as Silicibacter, potentially more sensitive to low concentrations of substrates. Finally, additional behavioural
strategies, such as quorum sensing, may allow some
strains of bacteria to enhance levels of accumulation following the initial detection of a diffusing nutrient
source.
Rather than simply reflecting the growth response of
bacteria to phytoplankton-derived DOC, it is becoming
evident that associations between phytoplankton and
heterotrophic bacteria are multi-faceted and complex
(Mayali and Azam, 2004). This is particularly true
within the context of HAB-forming toxic phytoplankton. The experiments presented here are the first to
directly demonstrate that the chemical products of the
toxic species H. akashiwo can provoke a strong behavioural response in some marine bacteria. This chemotactic behaviour may facilitate tight spatial coupling
between bacteria and H. akashiwo, which in the environment will have potential implications for the growth
and toxicity of this HAB forming species. These observations indicate the need for further in situ investigations
into the composition of bacterial communities occurring
in association with HAB forming phytoplankton and
the subsequent influence of bacterial populations on the
toxicity of species such as H. akashiwo.
treatments occurred in response to the chemical products of H. akashiwo.
These experiments indicate that the chemical products of the toxic phytoplankton H. akashiwo act as chemoattractants for some species of marine bacteria.
Bacterial chemotaxis to the chemical exudates of phytoplankton is widespread (Bell and Mitchell, 1972; Miller
et al., 2004; Stocker et al., 2008) and plays an important
role in aquatic microbial trophodynamics. Localized
associations between heterotrophic bacteria and phytoplankton allow bacteria to gain enhanced exposure to
the dissolved organic carbon (DOC) exuded by phytoplankton cells, while phytoplankton may receive
increased remineralized inorganic nutrients from the
bacteria (Azam and Ammerman, 1984). Alternatively,
increased exposure to algicidal strains of bacteria can
inhibit the growth of phytoplankton or kill them
(Mayali and Azam, 2004).
Pseudoalteromonas strains are often algicidal (Skerratt
et al., 2002; Mayali and Azam, 2004) and have been
shown to kill H. akashiwo (Lovejoy et al., 1998).
Furthermore, P. haloplanktis has been demonstrated to
markedly increase the toxic effects of H. akashiwo on fish
(Carrasquero-Verde, 1999). Hence, localized associations between P. haloplanktis and H. akashiwo may have
implications at several levels. However, while we have
previously found P. haloplanktis to exhibit strong chemotactic attraction to the exudates of diatoms, chlorophytes
and cyanobacteria (Stocker et al., 2008; Seymour et al.,
2008), no chemotactic response was observed here.
While the reason for this lack of attraction is not clear,
if P. haloplanktis has an algicidal effect on H. akashiwo
like other Pseudoalteromonas strains, it is plausible that
H. akashiwo may benefit from the production of chemicals that repel or inhibit the chemotaxis of this bacterium
(Strom, 2008). Alternatively, the chemical constituents of
H. akashiwo extracellular products may simply not match
the chemoreceptive capabilities of P. haloplanktis.
Vibrio alginolyticus exhibited the strongest attraction to
the H. akashiwo exudates. Interestingly, H. akashiwo has
been demonstrated to inhibit the growth of V. alginolyticus
through the production of reactive oxygen compounds
(Oda et al., 1997). Our results indicate that the
chemical products of H. akashiwo encourage attraction
by V. alginolyticus despite this potential inhibitory effect.
Silicibacter TM1040, like other members of the
Roseobacter clade, uses chemotaxis to form close spatial
associations with dinoflagellates (Miller et al., 2004) and
this behaviour significantly aids the growth of both the
bacterium and the associated phytoplankton (Miller and
Belas, 2006). We have also previously demonstrated that
TM1040 exhibits chemotactic responses to the exudates
of other phytoplankton species (Seymour et al., 2008).
AC K N OW L E D G E M E N T S
M. Polz, D.E. Hunt and R. Belas provided bacterial
strains.
FUNDING
This research was funded by NSF grant OCE-0526241
and OCE-0744641 CAREER to R.S., a Martin
Fellowship for Sustainability to T.A. and an Australian
Research Council Discovery Grant to J.R.S.
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15956) to low-molecular weight substances under starvation and
recovery conditions. Appl. Environ. Microbiol., 56, 3699– 3704.
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